The sensitivity of binding assays in which luminescent probes are used to detect a specific target of interest can be limited by high backgrounds from unincorporated or unbound probes. In heterogeneous assays, such as gel mobility shifts, backgrounds are reduced by physical separation of unbound probes from target-bound probes. However, this requires extra steps and individualized optimization of separation procedures depending on the targets to be analyzed. In some cases, inefficient (low) recovery of the separated targets can greatly decrease the detection sensitivity, requiring greater initial starting amounts of material. Many homogenous binding assays, which do not require separation of unbound from target-bound probes, rely on changes in fluorescence properties for detection. For example, some nucleic acid (NA) detection assays monitor changes in emission color ratios or intensities (fluorogenic polymerase chain reaction (PCR) assays, molecular beacons). While these types of NA detection approaches have helped to reduce backgrounds, they are limited, in part, by difficulties inherent in labeling and purifying dual labeled probes. Therefore, homogeneous assays would benefit from reagents that selectively reduce the emission of non-target bound probes while leaving target probe adducts luminescent.
Various concepts applying fluorescence quenching and energy transfer to reduce the fluorescence background have been applied to hybridization assays of PCR amplified targets, Morrison, L., “Homogeneous Detection of Specific DNA Sequences by Fluorescence Quenching and Energy Transfer,” 9 J. of Fluorescence, No. 3, pp. 187–196 (1999). Examples of these techniques are:
1. An adjacent probe format where a fluorescent labeled probe and a quencher probe are designed to bind to adjacent locations on a DNA target; the resulting proximity of the fluorescent label and the quencher act to reduce the fluorescence intensity from the bound fluorescent labeled probes so that a reduction in fluorescence intensity indicates the presence of the target.
2. A complementary probe pair is designed having complementary sequences, one with a fluorescent label and one with a quencher, where the single-stranded complementary probes compete for hybridization with each other and with single-stranded target DNA; the probes bind together in the absence of the target to quench fluorescence from the fluorescent label, but, in the presence of a target, one of the probes preferentially binds to the target sequence so that quenching is reduced.
3. In a variation of approach 1., above, a labeled primer is incorporated in one amplified DNA strand and subsequently hybridizes to a labeled probe that is complementary to the amplified sequence adjacent to the primer position to place the fluorescent label and quencher in an interaction distance to quench fluorescence when the target DNA is present.
4. In a variation of approach 2., above, the termini at one end of the probes are connected to form a hairpin that places the fluorescent label and quencher in a proximity to quench fluorescence in the absence of the target; upon binding to the target the hairpin opens, separating the quencher and the emitter whereby fluorescence from the emitter is observed.
5. In yet another approach, fluorescent-labeled probes and quencher probes are introduced prior to the start of PCR amplification so that DNA polymerase 5′-to-3′ exonuclease activity cleaves a single probe strand containing both fluorescent and quencher labels, thereby separating the quencher and emitter during the amplification process.
6. A multi-step PCR detection method is shown, where, following amplification, labeled primer, labeled dideoxynucleosidetriphosphates, and DNA polymerase are added to the reaction mixture. Labeled dideoxynucleotide becomes incorporated to indicate the presence of a particular target nucleotide at that location, thereby placing a second label within interaction distance with the first label of the primer strand.
In all of these cases, both the fluorescent-labeled probe and the quencher probe are present concurrently with the target NA, which increases the complexity of the design by requiring the same conditions for fluorescent labeling and quenching.
Various advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.